In the present study the histological changes in the various layers of the cornea were noted in various groups of buffaloes. The cornea of buffaloes was made up of five layers
i.
e. corneal epithelium, anterior limiting membrane or Bowman’s membrane, substantia propria or stroma, Descemet’s membrane and corneal endothelium (Fig 1).
Ageing changes in the corneal epithelium
The corneal epithelium of buffaloes was made up of non keratinized stratified squamous epithelium and consisted of 6-7 layers in 5 months of age (Fig 2), whereas, the number of layers gradually increased to 12-14 at the end of first year (Fig 3).
Dellmann and Eurell (2006) also reported 4-12 layers thick non keratinized stratified epithelium in domestic animals. In the present study three types of cellular layers were noted in the cornea without any distinct boundaries
viz., stratum basale, stratum intermedium and stratum superficial layers. The stratum basale contained low columnar to high cuboidal type during 5 months of postnatal age and they became high columnar type with advancement of age as reported by
Wiley et al., (1991) and
Li et al., (2007) in humans. The base and apex of these cells were narrow but they were bulged at the middle due to the presence of rounded nucleus. These cells were capable of mitosis besides the stem cells and transient amplifying cells and were the source of wing and superficial cells in all three groups of animals (Fig 3).
The intermediate zone cells (wing cells) were round to oval and their cytoplasm was more granular. These cells were present in 2-3 cell layers at 5 months of postnatal age, but they were in 6-7 layers with advancement of age. The nuclei of these were round to oval in shape and heterochromatic in all age group of animals (Fig 3). Similar findings were also reported by
Dellmann and Eurell (2006) in the cornea of adult domestic animals. But,
Del Monte and Terry Kim (2011) observed that these cells were flat and 2 to 3 cells thick in the cornea of humans.
The outer or superficial cells were elongated with tapered ends and arranged in 4 to 5 layers. They showed distinct nucleus with less cytoplasm. The shape of the nuclei in superficial cells of first group was flat and spindle shaped, but they became large and more elongated with advancement of age (Fig 3).
Kelly et al., (1971) also stated that none of the corneal cells lost their nuclei in normal keratinisation process in corneal epithelium of humans similar to the present observations in buffaloes.
Doughty et al., (1995) and
Farjo et al., (2008) reported that matted microplicae and apical microvilli on superficial corneal epithelial cells of humans, but in the present study no cellular modifications were observed on the superficial cells of the cornea in buffaloes (Fig 3).
In the present study numerous sub basal nerve plexuses were observed in the superficial stroma, immediately under the Bowman’s membrane. These plexuses were surrounded by numerous neurolemmocytes. The unmyelinated nerve fibers from these plexuses penetrated the Bowman’s membrane and reached the epithelium and formed sub epithelial nerve plexuses and they were distributed deeply between the basal, wing and superficial cells of corneal epithelium (Fig 4 and 5). Similarly,
Kelly et al., (1971), Schimmelpfennig (1982),
Muller et al., (1996), Oliveira-Soto and Efron (2001) and
Muller et al., (2003) also noted sub basal nerve plexus in superficial epithelium of cornea of humans in support of our present findings. Further, in the present study the density of nerve fibres in corneal epithelium of buffaloes was not altered with age, similarly
Erie et al., (2005) not reported any changes in distribution and density of nerve fibres in cornea of humans.
The pigmentation appeared sparsely between the basal cells and wing cells of the corneal epithelium in group I buffaloes, but it was increased from group II to group III buffaloes (Fig 6). This pigmentation was believed to be occurred from the dispersed melanocytes from the limbus. It was in accordance with the observations made by
Holloway (1969) in the corneal epithelium of old dogs and hogs.
The mean thickness (µm) of corneal epithelium in group I, II and III was 117.04±3.37, 122.59±2.23 and 133.71±3.55µm respectively. There was a significant (P<0.01) increase in thickness of epithelium of group III buffaloes when compared to group I and II (Table 1 and Fig 13).
Ageing changes in the anterior limiting membrane or Bowman’s membrane
A homogenous and non cellular Bowman’s membrane of cornea was noted below the basal layer of corneal epithelium (Fig 7). Contrary to this
Raghavan (1964) and
Banks (1993) stated that the Bowman’s membrane may not be distinct in different domestic animals. In the present study it was made up of predominately collagen fibres. Whereas,
Dellmann and Eurell (2006) noted more reticular fibers in Bowman’s membrane of domestic animals and
Mazher (2012) noted elastic fibers in goats.
In the present study the mean thickness (µm) of Bowman’s membrane was increased with advancement of age in buffaloes
i.
e. from group I to III animals (Fig 7).
Berlau et al., (2002) also reported an increase in thickness of Bowman’s membrane with advancement of age in humans. Contrary to this,
Jacobsen et al., (1984) reported a decrease in thickness of Bowman’s membrane with advancement of age in humans.
The mean thickness (µm) of Bowman’s membrane in group I, II and III was 12.97±0.86, 17.22±0.55 and 18.71±0.68 respectively. There was a significant (P< 0.01) increase in thickness of Bowman’s membrane of group III buffaloes when compared to group I and II (Table 1 and Fig 13).
Ageing changes in the substantia propria or stroma
The stroma of cornea was well developed and it constitutes the major component of the cornea in all age groups of buffaloes. The stroma was comprised of a uniform collagen fibril matrix with loosely arranged fibers in the rostral half and tightly packed in posterior half in early aged animals (Fig 8), whereas from 3rd year onwards to the group II and III animals the fibers were tightly packed in the entire stroma (Fig 9). Further, the stroma of cornea was different from other collagenous structures in its transparency, which was the result of precise organization of the stromal fibers and extracellular matrix.
Fini and Stramer (2005) and
Torricelli and Wilson (2014) described that these fibers were surrounded by specialized proteoglycans, keratin sulfate, chondroitin sulfate, dermatin sulfate side chains, which regulate hydration and structural properties of human eye.
The mean thickness (µm) of corneal stroma in group I, II and III was 600.75±10.33, 672.22±10.46 and 690.00±5.32µm respectively. There was a significant (P<0.01) increase in thickness of stroma of group III buffaloes when compared to group I and II (Table 1 and Fig 13). The increase in the total thickness of stroma of cornea between group I and group III animals was principally due to progressive increase in number of collagenous lamellae. According to
Daxer et al., (1998) the main factor causing increase in stromal thickness in adult humans was due to an increased collagen fibril diameter, axial period and intermolecular bragg spacing in the stroma of cornea. Pigmentation of the stroma was observed in aged buffaloes. This pigment represents the remnants of past metabolic activities of the stromal cells of the cornea as opined by
Bohnke and Masters (1999) in old aged humans. They also reported small highly reflective microdots in the stromal matrix of aged humans.
Keratocytes (Fibrocytes) were spindle shaped and interposed between the collagen fibers, which were elongated and comparatively thicker in the group I animals than the Group III animals. These cells were supposed to secrete collagen and matrix of the stroma.
Jester et al., (1999) and
Del Monte and Terry Kim (2011) noted that keratocytes were the major cells of the stroma, which maintain the integrity of this layer, producing collagen, glycosamino- -glycans and matrix metalloproteinases in humans as noted in the present study. The number and density of keratocytes were decreased with advancement of age in buffaloes
i.
e. from group I to III animals. Similarly,
Patel et al., (2001) also reported that keratocyte density was negatively correlated with increase in age and decreased 0.45 percent per year in humans. Similarly,
Berlau et al., (2002) and
Niederer et al., (2007) noted that keratocytes declined by 0.9% per year in the anterior stroma, 0.3% per year in the mid stroma and 0.3% per year in the posterior stroma in human with advancement of age. In the present study the nuclei of keratocytes were thick and spindle shaped in group I and they were thin in group II and III animals.
Ageing changes in the Descemet’s membrane
The Descemet’s membrane was a homogenous and non cellular fibrous band, plays a role in several important functions including collimation of light, endothelial cell differentiation, proliferation and structural integrity. It was predominately consisted of collagen fibers. It showed a dark anterior band zone and light posterior band zone in buffaloes (Fig 10).
Kato et al., (2003) and
Gottsch et al., (2005) also noted similar findings in humans. In the cornea of mice collagen fibers in Descemet’s membrane were mainly type IV, VIII, XVIII fibers and also non-collagenous components including fibronectin, laminin, nidogen, heparin sulfate and dermatin sulfate
(Hopfer et al., 2005). The thickness of anterior band zone (ABZ) was not altered from group I to group III animals, whereas the posterior band zone (PBZ) thickness was altered and increased with age advancement
i.
e. in group III animals (Fig 11 and 12) as reported by
Johnson et al., (1982) and
Murphy et al., (1984) in human. Further, they reported that over the ensuing decades of life, the ABZ remains well-demarcated and stable in thickness and appearance. In contrast, the posterior portion of the membrane directly subjacent to the endothelium progressive thickening of the PBZ contributed to the age-dependent growth of Descemet’s membrane from approximately 3 µm at birth to 5-6 µm at 20 years of age and to 13 µm at 80 years of age.
The mean thickness (µm) of Descemet’s membrane and endothelium together in group I, II and III animals was 16.48±0.91, 41.29±2.25 and 54.82±1.44 µm respectively. There was a significant (P<0.01) increase in thickness of Descemet’s membrane of group III buffaloes when compared to group I and II (Table 1 and Fig 13).
Ageing changes in the corneal endothelium
In buffaloes the corneal endothelial cells were squamous type and single layered. These cells were small and firmly adherent to each other and to the Descemet’s membrane. In histological profiles the cells were tightly packed and their nuclei were small and placed close to each other with little intervening cytoplasm in buffaloes of group I (Fig 11). The size of the corneal endothelial cells was increased with advancement of age, whereas the number of corneal endothelial cells decreased with advancement of age in buffaloes. In group II animals, the cells were further increased in size and greatly enlarged in group III buffaloes when compared with the group I. In group III animals, the intercellular space is more and the cell numbers was relatively less when compared with the young animals (Fig 12). These observations suggested that there was a regular degeneration of corneal endothelial cells and there was no replacement of the endothelial cells, leading to decrease in their population with advancement of age in buffaloes. It is in accordance with the findings of
Capella (1971) in humanbeings, he stated that the morphologic characteristics of the endothelium remain fairly consistent between the ages of 15 and 50 years, but the individual cells began to increase in size and showed an irregularity in their morphology at about 60- 65 years in humans
Jun et al., (2006) reported that in mouse central corneal endothelial cell density showed a rapid declined by 5232 ± 892 cells/mm2 at 2 weeks and 2532 ± 112 cells/mm2 at 16 weeks of age. Thereafter, cell density declined more slowly, reaching 2004 ±134 cells/mm2 at 24 months of age.
Gambato et al., (2014) reported in the cornea of humans as endothelial cell density decreased from approximately 3000–4000 cells/mm2 at birth to 2500 cells/mm2 in late adulthood in humans.
The total thickness (µm) of cornea in group I, II and III animals was 747.22±13.57, 853.33±11.05 and 897.22±8.06 µm respectively. These findings suggested that there was a significant (P<0.01) increase in total thickness of cornea with advancement of age in buffaloes (Table 1 and Fig 13) due to the enhancement of cellular and fibrous population in different layers of cornea.
